DHA-containing nutritional compositions and methods for their production

ABSTRACT

This invention provides a particulate material suitable for use as a nutritional supplement, particularly as an aquaculture feed. The particulate material has a high proportion of DHA residues in the lipid fraction, which may be up to 35% of the material, or even more. Preferably, the material has a mean particle size of from about 5 microns to about 10 microns. This invention also provides a method for preparing a particulate material suitable for use as an aquaculture feed from microbial biomass, preferably from algal cells having a high content of DHA residues, by obtaining a lipid fraction from the biomass, preferably by solvent extraction of broken cells, followed by separating a fraction containing phospholipids and proteins from the lipid fraction, and removing water from the protein/phospholipid fraction to form a low moisture particulate, preferably by spray-drying the protein/phospholipid fraction.

This application claim benefit to provisional application 60/054,563Aug. 1, 1997.

BACKGROUND OF THE INVENTION

The last decade has witnessed a decline in the resources of naturalfisheries that has now reached a level of global environmental crisis.Thirteen of the worlds seventeen major fisheries are classified by theUnited Nations Food and Agriculture Organization (F.A.O.) to be in perilor in steep decline. Even in the best case scenario of both maintainingthe fishery catch at a record 100 million metric tons and conservativepopulation growth, there will be a shortfall in global seafood supply bythe end of this decade for the first time in human history. Furthermore,per capita global seafood consumption is predicted to increase as theworlds population shifts during the next two decades from industrializedcountries to todays undeveloped and emerging economies; traditionallylarge consumers of seafood. The only way to meet the global demand forseafood by the year 2000 is if aquaculture replaces the insufficientnatural fisheries catch.

Expansion of the aquaculture industry requires that several significantproblems be addressed, and one of the most significant hurdles toestablishing and maintaining an economically viable aquacultureoperation is the difficulty of supplying nutritionally balanced feeds.Larval fish, bivalves and crustaceans raised in the wild consume a mixedpopulation of feed organisms that collectively provide balancednutrition. On the other hand, fish larvae, bivalves and crustaceansraised in aquaculture farms can be difficult to rear and require livefeeds (algae or algae-fed rotifers and Artemia) for their nutrition.These live feeds are difficult to produce and maintain, require highlabor inputs and specialized facilities, and as a result larval feedsconstitute a significant cost to the aquaculture industry.

It is important that aquaculture feeds be nutritionally balanced so thatthe larvae receive proper nutrition. DHA (docosahexaenoic acid) has beenidentified as an important nutrient that contributes significantly tolarval growth and survival (Fulks, W and KL Main (eds). 1991. “Rotiferand Microalgae Culture Systems. Proceedings of a US-Asia Workshop.”Honolulu, Hi. The Oceanic Institute. Tamaru, CS, CS Lee and H Ako, 1991.“Improving the larval rearing of striped mullet (Mugil cephalus) bymanipulating quantity and quality of the rotifer, Brachionusplicatilis.” In: W. Fulks and KL Main (eds), 1991). Larvae ultimatelyacquire these fatty acids from algae, either by directly feeding onalgae with high levels of polyunsaturated fatty acids or by feeding onrotifers and Artemia that have been reared on algae high inpolyunsaturated fatty acids. Unfortunately, the algae, artemia androtifers used at aquaculture farms are low in DHA, reducing the survivalrates for the larvae below their maximal rate and increasing the cost ofthe final aquaculture farm product. If sufficient DHA could be providedto the larvae it is expected that the survival rate for larvae wouldincrease, thus reducing the cost of farm-raised seafood.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a particulate materialsuitable for use as an aquaculture feed. The particulate material has ahigh proportion of DHA residues in the lipid fraction, which may be upto 35% of the material. Preferably, the material has a mean particlesize of from about 5 microns to about 10 microns.

In another embodiment, this invention provides a method for preparing aparticulate material suitable for use as an aquaculture feed frommicrobial biomass, preferably from algal cells having a high content ofDHA residues, by obtaining a lipid fraction from the biomass, preferablyby solvent extraction of broken cells, followed by separating a fractioncontaining phospholipids and proteins from the lipid fraction, andremoving water from the protein/phospholipid fraction to form a lowmoisture particulate, preferably by spray-drying theprotein/phospholipid fraction.

In still another embodiment, this invention provides a particulatematerial containing phospholipids having docosahexaenoic acid (DHA)residues, the particulate material being prepared by drying a slurrycomprising a polar lipid extract from DHA-containing microbes, and dry,particulate material may be prepared from a slurry which issubstantially free of material that did not come from the DHA-containingmicrobes, particularly by spray drying the slurry. Typically, at leasttwo thirds of the dry matter in the polar lipid extract ofDHA-containing microbes is material derived from the microbial cells;preferably less than 25% of the dry matter is non-microbial, morepreferably less than 20%, even more preferably 15% or less. Typically,the spray dried particulate has number average particle size between 5microns and 10 microns.

In yet another embodiment, this invention provides a method forpreparing a DHA-containing particulate material comprising (a) lysingDHA-containing microbial cells; (b) extracting lysed cells with solvent;(c) separating a polar lipid fraction from the extract; and (d) dryingthe polar lipid fraction, with or without addition of other nutrients,to form a particulate material. Preferably, the polar lipid fraction isan aqueous slurry which is dried by spray drying.

In a particular embodiment, this invention provides a method forpreparing a DHA-containing particulate material comprising drying aslurry containing polar lipids extracted from dinoflagellates, whereinthe dried material is in the form of particles having a mean particlediameter between 5 and 10 microns.

In yet another embodiment, this invention provides an aqueous emulsionor suspension containing phospholipids with DHA residues obtained from apolar lipid extract from DHA-containing microbes. Preferably, at least10% of the fatty acid residues in lipids of the DHA-containing microbesare DHA residues. More preferably, at least 10% of the fatty acidresidues in polar lipids of the DHA-containing microbes are DHAresidues.

In still another embodiment, this invention provides a compositioncomprising a particulate material containing phospholipids with DHAprepared by drying a slurry comprising (a) a polar lipid extract fromDHA containing microbes and (b) a meal containing protein and/orcarbohydrate. Preferably, the meal comprises microbial cells or cellfragments, which may be cells or cell fragments which have beenextracted to remove part of the lipids, or even most of the lipids.Preferred microbial cells or cell fragments are from Chlorella,Crypthecodinium, or a yeast such as Saccharomyces, or a fungus, such asMorteriella, Schizochytrium, or Thraustochytrium.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Producing high DHA-containing oil from biomass requires severaldifferent processing and refining steps. Each refining step produceswaste fractions as a byproduct of the oil purification. These byproductscontain significant levels of DHA and other nutrients that can havepotential use as aquaculture feeds for fish larvae, bivalves andcrustaceans.

One byproduct in particular contains a very high level of DHA as well asother nutrients (carbohydrate, protein, etc) that are useful andvaluable in larval nutrition. This byproduct, hereinafter called “DHAPhospholipids,” also contains significant levels of all of the aminoacids that are considered essential for larvae. The ten amino acids thatare generally recognized to be essential for most fish species arehistidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan and valine, see, e.g., Shepherd, Jonathan andNiall Bromage, Intensive Fish Farming, Chapter 5, Nutrition and Growth,Blackwell Scientific Publications, Boston, 1992, pp. 154-197. The DHAPhospholipid material contains all of these amino acids at levelscomparable to the levels found in some other aquaculture feeds. The DHAPhospholipid material contains phospholipids having a high proportion ofDHA residues, as well as other lipid components such as triglycerides,free fatty acids, sterols, glycolipids, etc. The biochemical compositionof the DHA Phospholipids, obtained as spray-dried particles, is given inTable 1. The fatty acid composition of the particulate material is givenin Table 2.

Source of DHA-containing Material

The principal source of DHA in the biosphere is from algae. Numerousalgal species produce DHA. One species in particular (Crypthecodiniumcohnii) has been manipulated to produce very high levels of DHA. Thisorganism has been cultivated at large scale and the biomass has beenused for the production of a high DHA-containing oil. Suitable processesfor producing DHA-containing biomass from C. cohnii are provided in U.S.Pat. Nos. 5,397,591 and 5,492,938, incorporated herein by reference.

Similar DHA Phospholipid material may be made from any single celledorganism that contains a significant amount of DHA This would includevarious oleaginous fungi, various algae (especially members of the classDinophyceae, Bacillariophyceae, Chlorophyceae, Prymnesiophyceae andEuglenophyceae), as well as organisms of uncertain taxonomic status suchas Thraustochytrium or Schizochytrium. Suitable processes for producingDHA-containing biomass from Thraustochytrium or Schizochytrium areprovided in U.S. Pat. No. 5,130,242, incorporated herein by reference.

Typically, microbes serving as a source of DHA Phospholipids accordingto this invention will have at least 5% of the fatty acids and fattyacid residues in the microbe as DHA, more typically at least 10%, andpreferably at least 20%. Preferably, at least 10% of the fatty acidresidues in the polar lipids of the source microbe will be DHA, morepreferably at least 20% of the residues will be DHA.

Preparation Methods

DHA Phospholipids may be obtained from a lipid fraction of theDHA-containing biomass. A preferred method for obtaining a suitablelipid fraction is solvent extraction of lysed cells. In a preferredmode, the biomass may be spray-dried, which will lyse most cells, andthe spray-dried material extracted with a solvent such as hexane.Grinding or homogenizing the solvent-biomass mixture may facilitateextraction of the lipid fraction (oil) and may also promote recovery ofnon-lipid components that are desirable for the aquaculture feed. TheDHA Phospholipid material is separated from the oil as awater/salt-containing precipitate. Suitable procedures for suchseparation are readily adapted from analogous processes in vegetable oilrefining.

Although the DHA Phospholipid byproduct contains useful levels of DHAand other nutrients, a suitable method must be found to provide thismaterial to larvae. Many fish larvae require live feed, and bivalves andcrustaceans also require either live feed organisms or particles of alimited size range. Thus, further processing is preferable to merelyproviding this material directly to the fish larvae, bivalves orcrustaceans.

For those organisms requiring a live feed, the DHA Phospholipids can befed to arterria and/or rotifers (both natural food organisms forlarvae), with the goal of raising the DHA level of the artemia/rotifersand subsequently raising the DHA level of the larvae. For thoseorganisms that are filter feeders, the material may be supplied as smallparticles and thus provide a direct enrichment of DHA into the diet.

In order to prepare particles of the proper size for ingestion byartemia, rotifers and filter feeders, the DHA Phospholipid material isdried by any suitable process, such as spray drying, granulation,encapsulation and the like. When DHA Phospholipid material was freezedried, the resultant particles were too large to be useful. The high fatcontent in the freeze-dried material did not permit the particle size tobe reduced by grinding. Spray drying yielded a crumbly mass consistingof particles that were of an appropriate size range for consumption byartemia, rotifers and filter feeders. Mean particle size was determinedto be about 5-10 microns in diameter; the distribution of partical sizesabout the mean was quite broad ( at least from >30 microns to <1micron). Although the material was subjected to heat during the dryingprocess, the DHA level was not appreciably reduced by the temperature ofthe dryer. Thus, spray drying yielded a product with a high DHA contentand an appropriate particle size.

Preferably, the DHA level of the spray dried material should bemaximized, thus making the material an even more valuable source of DHAfor larvae. Typically, the DHA level would be raised by increasing theamount of DHA-containing oil in the DHA Phospholipid material, which maybe accomplished by adding refined triglyceride oil, partially purifiedtriglycerides or crude lipid extract from a microorganism that is highin DHA. The inventor has noted that, when the triglyceride level(measured as esterified fatty acid level) exceeded about 30% of thetotal dry weight of the DHA Phospholipid, the material did not spray dryas well and appeared to form very large particles that were lesssuitable for this application. At triglyceride contents above 40%, thefinal spray-dried product is very sticky due to the high fat content,which may explain the apparent high particle size. This material is notas easy to handle as the lower triglyceride material, but it still canbe used as an artemia and rotifer enrichment product. Of course,supplemental DHA may also be supplied from non-microbial sources,including purified DHA-alkanol esters, but these sources may be lesseconomical.

While DHA Phospholipid may be spray dried directly, spray drying may befacilitated if the slurry contains typical spray drying agents when itis fed into the spray dryer. Suitable spray drying carriers includemaltodextrines, starch, gelatin, sugars, and molasses. Preferredcarriers include sodium alginate and gum arabic. In addition, variousadditives whose presence in the final particulate is desirable fornutritional reasons may also be added to the slurry before drying, suchas proteins, carbohydrates, fatty acids and lipids, or vitamin sources,including additives like yeast extract, starch, gelatin, gluten, etc. Inaddition, amino acids may be added to the slurry, such as methionine, aparticularly important amino acid in fish nutrition. Alternatively,algal biomass may be added to the slurry as a spray drying aid,preferably using algae which are naturally food for rotifers or fishlarvae.

In one embodiment, particulate material may be prepared by formulatingand spray drying the DHA Phospholipids with microbial biomass, which maybe whole or lysed cells or cellular material after lipid extraction. DHAPhospholipids may be mixed with biomass in any ratio, including 1:100 to100:1, preferably in ratios from 1:10 to 10:1. The mixture is typicallyspray dried from an aqueous slurry to form a material with theappearance or consistency of cornmeal. Typically, the biomass will befrom microbes that have known nutritional uses, such as Chlorella.Chlorella is consumed as a human food product, and DHA is an importanthuman nutrient, so such particulate material is also useful in humannutrition. Of course particulate material produced according to thisinvention may be used to supply DHA in human nutrition whether or notthe particulate contains Chlorella, so long as the process conditionsand any other additives included in the slurry are suitable for humanconsumption.

While the DHA Phospholipid is particularly suited to spray drying,particulate material containing high DHA levels may also be preparedaccording to this invention by granulation or encapsulation. Conditionsfor granulation or encapsulation of DHA Phospholipid may be readilyselected by the skilled worker, typically using one or more of theadditives which are listed above for use in spray drying, such asalginate or gum acacia. Of course, DHA Phospolipids may also be coatedon oilseed meal (e.g., soybean meal, cotton seed meal, corn germ, etc.).Furthermore, mixtures of DHA Phospholipids with solvent extracted algalbiomass (“biomeal”) are also suitable for freeze-drying. While thisinvention is particularly suitable for producing dry products enrichedin DHA, the DHA Phospholipids obtained from microbes according to thisinvention may be formulated in a suspension or emulsion for subsequentuse, particularly as a nutritional supplement. Such emulsions orsuspensions containing polar lipids may be routinely prepared, as isknown in the art.

Particle size distribution in the dried material may be adjusted byfractionation in, e.g., cyclones or filter bags, to remove particlesthat are too large, but preferably the drying process will produce aparticulate material of mean particle size from 5 microns to 10 microns.Typically the dried DHA Phopholipids will be mixed with sea water beforefeeding to larvae, rotifers, or artemesia. Varying the agitation in thismixing step will permit post-drying adjustment of the particle size.

Another fatty acid with significance in both aquaculture and human andanimal nutrition is arachidonic acid (AA). Arachidonic acid may also beobtained in lipid extracts of microbes, although preferred sources of AAare different species from the preferred sources of DHA. Suitablemicrobial sources of AA and methods for growing them are taught, forexample, in U.S. Pat. No.5,658,767. Extraction of crude lipids fromAA-containing microbes leaves a protein-and-carbohydrate containingresidue that may also still have appreciable amounts of AA as fattyacids and/or fatty acid residues. This material may be referred to as AAbiomeal.

During the processing and refining of the AA-containing crude lipidextract, several fractions are produced, including AA Phospholipids. TheAA Phospholipids can also be blended with the DHA Phospholipids toproduce a material that contains both fatty acids in the ratio that willprovide for optimal nutrition. Such a blend can be prepared as a drymaterial or as an emulsion or suspension. The AA Phospholipids canalso-be blended with the DHA biomeal and dried or emulsified to producea material that is high in arachidonic acid. Through the use ofdifferent ratios of DHA Phospholipids, DHA biomeal, AA biomeal, and/orAA Phospholipids, a material can be produced that has the appropriateratio of DHA and AA for optimal nutrition.

EXAMPLES

In order to facilitate a more complete understanding of the invention, anumber of Examples are provided below. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only.

Example 1 Process for Producing DHA Phospholipids

Crypthecodimium cohnii is grown by heterotrophic fermentation asdescribed in U.S. Pat. No. 5,407,957, incorporated herein by reference.At the conclusion of the fermentation, the DHA-rich biomass isconcentrated by centrifugation to about 18% solids (range is 5-35%solids). The concentrated biomass is held in a chill tank (50-60° F.)prior to spray drying. The material is pumped under high pressurethrough a spray dryer with an inlet temperature ranging between 300-400°F., but typically 350-380° F. The flow rate into the spray dryer isadjusted as necessary to achieve an outlet temperature of 180-240° F.,but typically 205-215° F. The dried biomass recovered from the spraydryer has a moisture content ranging from 1-15% (typically 6%).

The dried biomass is slurried with hexane at the ratio of about 2 litersof hexane per kilogram of dried biomass. The hexane/biomass mixture maybe milled in a colloid mill to insure that all cells are broken and thatmaximum oil can be extracted. Alternatively, the biomass/hexane mixturemay be homogenized (e.g., forced through an oriface with pressure dropof approximately 7-800 atm). The hexane fraction (containing theDHA-rich oil) is separated from the solids using a counter currentextractor. The hexane exiting from the extractor has an oil content ofabout 5% (v:v).

The oil/hexane mixture is centrifuged to remove any fine particles thatwere not previously removed in the extractor. The hexane is partiallyremoved by evaporation to produce a mixture of about 65% oil in hexane.This oil/hexane mixture is cooled to about 40° F. and held at thistemperature for about 12 hours. The material that precipitates under thelow temperature condition is removed by centrifugation. The remaininghexane associated with the oil is removed by evaporation to produce ahexane-free oil.

The oil is transferred to a refining tank where 50% caustic (0.74% w/wbased on oil), 85% phosphoric acid (0.2% w/w based on oil), and oleicacid (to bring the final free fatty acid level to 5% in the oil) areadded to the crude oil. The mixture is agitated and heated toapproximately 70° F. for 15-30 minutes. The combination of heat andchemicals causes the DHA Phospholipids to precipitate out of the oil.The DHA Phospholipids are collected by centrifugation.

The DHA Phospholipid fraction is homogenized to insure a uniformmixture. At this point the material can be used “as is” for spray dryingor it can be blended with various nutrients and other compounds (i.e.vitamins, protein, alginates, antifoams, etc.) before proceeding withspray drying. The solids content of the DHA Phospholipids can range from10% up to over 80%. Typically, the solids content is diluted to around25-35% to form a pumpable slurry for spray-drying, although solidscontent in the slurry may range from 10-50%. The material is spray driedusing either a high pressure injection nozzle or a rotary atomizer.Suitable inlet temperatures range between 170-240° F., but typicallyaround 200° F., with the material being fed to the spray dryer at a ratesufficient to maintain an outlet temperature on the spray dryer of150-200° F., but typically between 180-190° F. In an alternative mode,the slurry is spray dried with inlet temperature of about 300° C. (540°F.) and outlet temperature of about 110° C. (230° F.). Routineoptimization of drying conditions by adjustment of inlet temperature andflow rate in view of the guidelines provided herein is contemplated forthis invention.

The DHA Phospholipid material collected from the spray dryer has amoisture content of about 4%, and the biochemical composition of threerepresentative batches is given in Table 1, the fatty acid compositionof the same 3 batches, plus a 4th is given in Table 2. These batcheswere all suitable for spray drying. If the esterified fatty acid levelsexceed about 30-35%, then the material does not spray dry as well.Rather than forming discrete particles, it tends to form larger clumps,presumably due to the higher oil content in the material.

Example 2 DHA-containing Particulate Blends

The DHA Phospholipids may be formulated with other materials andnutrients. Table 3 compares spray dried DHA Phospholipids to thecomposition of DHA Phospholipids+Biomeal and DHA Phospholipids+Chlorellaprepared as follows:

a. DHA-Phospholipids was mixed with biomeal consisting of dried biomassfrom Example 1 after extraction with solvent, and then spray dried asdescribed in Example 1. The purpose of this blending was to reduce thetotal fatty acid content of the spray dried material so that it wouldn'tbe so sticky. This blend was certainly less sticky so it may beconsidered an improvement to the basic material, even though blending inthe biomeal did reduce the total fat content compared to the basic DHAPhospholipids.

b. The DHA Phospholipids were blended with the alga Chlorella, and thismixture was spray dried as described in Example 1. Chlorella is anatural food source for rotifers and is high in protein. By blendingwith Chlorella it was possible to reduce the stickiness of the finalspray dried DHA Phospholipids while at the same time providing nutrientsfrom the natural food source. The initial trial was to blend 3:1 (w:w)DHA Phospholipids: Chlorella, and this produced a material that handledvery well. A wide ratio of blends is possible. One variation is to havea high ratio of Chlorella to DHA Phospholipids. This would basicallyprovide the natural marine food source with extra DHA, so the materialcould be as a regular, routine feed and not just as an enrichment toincrease the DHA content of the rotifers prior to feeding them to fishlarvae.

c. The DHA Phospholipids have also been mixed with a protein source forthe purpose of improving the protein content (and therefore nutritionalcontent) of the spray dried material. Specifically, the DHA Phospholipidwas blended with soy protein hydrolyzate in a ratio of 3:1 (w:w) ofDHA-Phospholipid: soy protein hydrolyzate. This demonstrates that theDHA Phospholipid can be mixed with other nutrients to make aparticulate.

Example 3 Blends of DHA and Arachidonic Acid Materials

The DHA Phospholipids can be formulated with arachidonic acid to producea material that provides both of these fatty acids in a single nutritionproduct. Arachidonic acid-containing biomass is produced fromMorteriella as outlined in U.S.Pat. No. 5,658,767. The Morteriellabiomass is harvested by centrifugation and dried under vacuum. The driedbiomass is extracted with hexane to remove the arachidonicacid-containing lipids, and the crude lipid fraction is processed in amanner similar to that used in Example 1. The mass remaining after lipidextraction removal (referred to as AA biomeal) contains a high level ofprotein but also significant amounts of arachidonic acid. This AAbiomeal can be blended with the DHA Phospholipids to produce a higherprotein material that also contains significant amounts of botharachidonic acid and DHA.

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in edible oilextraction and processing, microbial fermentation, nutrition, medicine,pharmacology, and/or related fields are intended to be within the scopeof the invention, which is limited only by the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

TABLE 1 Biochemical composition of spray dried DHA PhospholipidsComponent Prep. 1 Prep. 2 Prep 3 Total Fatty Acid 57.4 61.3 55.6 (% oftotal wt) esterified fatty acid 25.6 24.4 22.3 (% of total wt) (48% DHA)(47.7% DHA) (47.9% DHA) free fatty acid 31.8 36.9 33.3 (% of total wt)(19% DHA) (22.5% DHA) (25.0% DHA) Sterol (% of total wt) Totalcarbohydrate 8.0 7.2 8.3 (% of total wt) glucose 6.1 4.7 6.2 galactose1.9 2.5 2.1 fucose 0.0 0.0 0.0 Protein (% of total wt) 13.2 9.7 11.7Amino Acid (% of protein) methionine 2.0 4.3 2.23 cystine 0.8 1.1 0.75lysine 4.3 3.7 4.92 phenylalanine 5.0 5.0 1.73 leucine 8.3 8.2 7.54isoleucine 4.0 4.2 4.65 threonine 5.9 5.4 5.89 valine 7.2 5.4 6.99histidine 2.5 2.2 2.56 arginine 4.8 4.3 4.74 glycine 6.2 6.2 7.05aspartic acid 10.4 10.3 10.66 serine 5.5 6.0 6.26 glutamic acid 13.713.8 14.06 proline 6.2 4.7 6.77 hydroxyproline 0.5 1.7 0.00 alanine 7.68.0 8.81 tyrosine 3.8 4.3 2.94 tryptophan 1.4 1.2 1.46 Moisture (% oftotal wt) 4.4 4.00 6.0 Fiber (% of total wt) 0.7 1.0 0.6 Ash (% of totalwt) 10.4 10.1 10.6 sodium (% of total wt) 2.6 not analyzed 3.38 TOTAL94.1 93.3 92.7

TABLE 2 Fatty acid composition of spray dried DHA Phospholipids (% oftotal fatty acid) Prep. 1 Prep. 2 Prep. 3 Prep. 4 Prep. 1 non- Prep. 2non- Prep. 3 non- Prep .4 non- Fatty Acid esterified esterifiedesterified esterified esterified esterified esterified esterified 08:000.06 0.25 0.23 10:00 0.67 1.22 0.83 1.11 0.45 0.76 1.39 0.5 11:00 12:004.34 2.69 4.36 2.65 3.34 3.14 5.71 2.71 13:00 14:00 15.94 5.81 15.52 7.714.83 9.68 16.71 8 14:01 0.13 0.12 0.11 16:00 17.2 11.77 16.21 13.6116.45 15.26 20.8 16.34 16:01 1.7 0.47 1.63 0.6 1.4 0.53 1.16 0.46 16:0216:03 17:00 0.13 18:00 0.88 1.89 0.94 1.91 0.96 1.82 1.11 1.68 18:1n-910.21 46.29 12.82 40.61 13.29 36.1 9.22 36.97 18:1n-7 0.62 0.63 0.460.56 18:02 0.1 8.56 7.47 0.4 6.06 0.1 7.3 18:3n-6 0.27 18:3n-3 0.12 0.1118:04 20:00 0.09 0.32 0.26 0.09 20:01 0.15 0.13 20:02 20:3n-9 20:3n-60.3 0.2 20:3n-3 0.61 20:04 20:05 22:00 22:01 22:04 0.46 0.42 22:5n-60.64 22:5n-3 0.53 0.28 0.28 22:06 48.05 19.62 47.68 22.52 46.96 25.0142.85 22.78 % Fatty acid in sample 25.64 31.83 24.4 36.9 22.34 33.2631.2 45.63 % Unknown 0.23 0.41 0 0.26 0.23 0.71 1.09

TABLE 3A COMPARISON OF AQUACULTURE PRODUCTS DHA DHA-PL + DHA-PL +Category Phospholipids C.cOHNII Chlorella Total Fatty 76.8 57.3 56.7Acid (%)  DHA (% of fat) 30.9 32.91 30.54  DHA (% DW) 23.7 18.9 17.3 EPA (% of fat) 0.11 0 0 Carbohydrate 2.1 18.1 9.7 Protein (%) 11.7 10.2Moisture (%) 1.5 2.4 Ash (%) 9.3 10.8 Fiber (%) 1 0.8 Total 102.4 97.2

TABLE 3B FATTY ACID ANALYSIS Fatty Acid DHA DHA-PL + DHA-PL + (% oftotal fatty acid Phospholipids Ccohnii Chlorella 08:00 0.24 0.17 0.0610:00 0.86 0.64 1.06 11:00 0.06 12:00 3.93 3.41 3.23 13:00 0.00 14:0011.54 12.05 8.61 14:01 0.04 0.06 0.05 16:00 18.15 16.58 12.49 16:01 0.740.96 1.18 16:02 0.12 16:03 0.08 17:00 18:00 1.45 1.42 1.87 18:1n-9 25.726.93 33.88 18:1n-7 0.33 0.32 0.46 18:02 4.38 4.58 7.46 18:3n-6 18:3n-30.07 0.30 18:04 20:00 0.05 20:01 0.08 20:02 20:3n-9 20:3n-6 20:3n-3 0.3620:04 20:05 22:00 22:01 22:04 0.25 22:5n-6 22:5n-3 0.11 22:06 30.9332.91 28.85 Total 99.22 100.03 99.76 (% fat in fatty acids) % Fatty acidin sample 76.8 57.30 56.70 % Unknown

What is claimed is:
 1. A particulate material containing phospholipidswith docosahexaenoic acid (DHA) residues prepared by drying a slurrycomprising a polar lipid extract from DHA-containing microbes.
 2. Theparticulate material of claim 1, wherein the mean particle size isbetween 5 microns and 10 microns.
 3. The particulate material of claim1, wherein the slurry is dried by spray drying.
 4. The particulatematerial of claim 1, wherein the slurry is substantially free ofmaterial which did not originate in said DHA-containing microbes.
 5. Theparticulate material of claim 1, wherein at least 10% of the fatty acidresidues in lipids of said microbes are DHA residues.
 6. The particulatematerial of claim 1, wherein at least 10% of the fatty acid residues inpolar lipids of said microbes are DHA residues.
 7. The particulatematerial of claim 1, wherein said microbes are dinoflagellates.
 8. Theparticulate material of claim 1, wherein said microbe areCrypthecodinium cohnii.
 9. A method for preparing a DHA-containingparticulate material comprising drying a slurry containing polar lipidsextracted from dinoflagellates, wherein the dried material is in theform of particles having a mean particle diameter between 5 and 10microns.
 10. A method for preparing a DHA-containing particulatematerial comprising lysing DHA-containing microbial cells; extractinglysed cells with solvent; separating a polar lipid fraction from theextract; and drying the polar lipid fraction, with or without additionof other nutrients, to form a particulate material.
 11. The method ofclaim 10, wherein the polar lipid fraction is an aqueous slurry which isdried by spray drying.
 12. The method of claim 10, wherein the microbialcells are dinoflagellate cells.
 13. The method of claim 10, wherein themicrobial cells are cells of Crypthecodinium cohnii.
 14. An aqueousemulsion or suspension containing phospholipids with docosahexaenoicacid (DHA) residues prepared by Homogenizing with water a polar lipidextract from DHA-containing microbes.
 15. The emulsion or suspension ofclaim 14, wherein at least 10% of the fatty acid residues in lipids ofthe microbes are DHA residues.
 16. The emulsion or suspension of claim14, wherein at least 10% of the fatty acid residues in polar lipids ofsaid microbes are DHA residues.
 17. The emulsion or suspension of claim14, wherein said microbes are dinoflagellates.
 18. The emulsion orsuspension of claim 14, wherein said microbes are Crypthecodiniumcohnii.
 19. A composition comprising a particulate material containingphospholipids with DHA prepared by drying a slurry comprising a polarlipid extract from DHA-containing microbes and a meal containingprotein, carbohydrate, or both.
 20. The composition of claim 19, whereinmeal comprises microbial cells or cell fragments.
 21. The composition ofclaim 19, wherein the microbial cells or cell fragments are fromChlorella.
 22. The composition of claim 19 wherein the microbial cellsor cell fragments are from Crypthecodinium.
 23. The composition of claim19, wherein the microbial cells or cell fragments are from a yeast. 24.The composition of claim 19, wherein the microbial cells or cellfragments are from Morteriella.
 25. A method of aquaculture comprisingfeeding particulate material containing a polar lipid extract frommicrobes comprising phospholipid with DHA residues to live larval feedorganisms comprising artemia, rotifers, or a combination thereof toenrich DHA level in the larval organisms; and feeding DHA-enriched livelarval organisms to fish larva, bivalves, crustaceans, or a combinationthereof.
 26. A method of aquaculture comprising feeding particulatematerial containing a polar lipid extract from microbes comprisingphospholipid with DHA residues to bivalves and/or crustaceans.
 27. Themethod of claim 25 or 26, wherein particulate material containingphospholipid with DHA residues has mean particle size from about 5microns to about 10 microns.
 28. The method of claim 25 or 26, whereinparticulate material containing phospholipid with DHA residues comprisesDHA and EPA in ratio of at least 300:
 1. 29. The method of claim 25 or26, wherein particulate material containing phospholipid with DHAresidues further comprises vitamins, amino acids, or both.
 30. Themethod of claim 25 or 26, wherein particulate material containingphospholipid with DHA residues further comprises Chlorella biomass. 31.The method of claim 25 or 26, wherein particulate material containingphospholipid with DHA residues is prepared by spray-drying aphospholipid-containing byproduct produced in refining a lipid extractfrom microalgae.